1. Field of the Invention
The present invention relates generally to various outer membrane proteins (OMPs) of Moraxella catarrhalis for use as targets in immunotherapy, such as in the preparation of vaccines or protective antibodies for use in treatment of Moraxella catarrhalis-related diseases. In particular aspects, the present invention concerns antigens identified by molecular weights of about 30 kD, 80 Kd (CopB) and a third antigen, termed "high molecular weight protein" or "HMWP" antigen having a molecular weight of between about 200 and 700 kD, as measured by SDS-polyacrylamide gel electrophoresis. In other aspects, the invention concerns recombinant clones and DNA segments encoding M. catarrhalis antigens and fragments and equivalents thereof, as well as to antibodies reactive with these species. Further, the invention concerns methods for the detection of M. catarrhalis antigens and antibodies, as well as the use of specific antigens and antibodies both in passive and active immunity against M. catarrhalis infections.
2. Description of the Related Art
It was previously thought that Moraxella catarrhalis (previously known as Branhamella catarrhalis or Neisseria catarrhalis) was a harmless saprophyte of the upper respiratory tract (Catlin, 1990; Berk, 1990). However, during the previous decade, it has been determined that this organism is an important human pathogen. Indeed, it has been established that this Gram-negative diplococcus is the cause of a number of human infections (Murphy, 1989). M. catarrhalis is now known to be the third most common cause of both acute and chronic otitis media (Catlin, 1990; Faden et al., 1993;1991; Marchant, 1990), the most common disease for which infants and children receive health care (Consensus, 1989). This organism also causes acute maxillary sinusitis, generalized infections of the lower respiratory tract (Murphy & Loeb, 1989), and is an important cause of bronchopulmonary infections in patients with underlying chronic lung disease and, less frequently, of systemic infections in immunocompromised patients (Melendez & Johnson, 1990; Sarubbi et al., 1990; Schonheyder & Ejlertsen, 1989; Wright & Wallace, 1989).
The "Consensus" report referred to above concluded that prevention of otitis media is an important health care goal due to both its occurrence in infants and children, as well as certain populations of all age groups. In fact, the total financial burden of otitis media has been estimated to be at least 2.5 billion annually, or approximately 3% of the health care budget. Vaccines were identified as the most desired approach to the prevention of this disease for a number of reasons. For example, it was estimated that if vaccines could reduce the incidence of otitis media by 30%, this outcome could bring about an annual health care savings of at least $400 million. However, while some progress has been made in the development of vaccines for 2 of the 3 common otitis media pathogens, Streptococcus pneumoniae and Haemophilus influenzae, there is no indication that similar progress has been made with respect to M. catarrhalis. This is particularly troublesome in that M. catarrhalis now accounts for approximately 17-20% of all otitis media infection (Murphy, 1989).
Previous attempts have been made to identify and characterize M. catarrhalis antigens that would serve as potentially important targets of the human immune response to infection (Murphy, 1989; Goldblatt et al., 1990; Murphy et al., 1990). Generally speaking, the surface of M. catarrhalis is composed of outer membrane proteins (OMPs), lipooligosaccharide (LOS) and fimbriae. As Murphy points out, M. catarrhalis appears to be somewhat distinct from other gram-negative bacteria in that attempts to isolate the outer membrane of this organism using detergent fractionation of cell envelopes has generally proven to be unsuccessful in that the procedures did not yield consistent results (Murphy, 1989; Murphy & Loeb, 1989). Moreover, preparations were found to be contaminated with cytoplasmic membranes, suggesting an unusual characteristic of the M. catarrhalis cell envelope.
More recently, isolation procedures have been reported for obtaining M. catarrhalis outer membrane components which result in what are said to be less-contaminated membrane preparations (Murphy & Loeb, 1989). Although this has allowed M. catarrhalis outer membrane protein profiles to be assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), these techniques did not lead to the isolation or characterization of OMPs (Murphy & Loeb, 1989). Indeed, information regarding individual OMPs is still limited to their apparent molecular weight on SDS/PAGE, with protein bands on SDS gels being grouped into general classes (Murphy & Loeb, 1989; Murphy, 1989). Seven or eight major OMP groups have been identified in this manner and appear to be fairly consistent between diverse M. catarrhalis strains. For example, OMPs have been grouped into classes A-H, beginning with bands of molecular weight around 98 kD (OMP-A) and proceeding to bands with molecular weights of about 21 kD (OMP-H) (Murphy & Loeb, 1989; Murphy, 1989).
The LOS of M. catarrhalis has also been suggested as a possible target for vaccine development. LOS has been isolated from M. catarrhalis strains and subjected to SDS-PAGE and silver staining (Murphy, 1989). In common with the OMPs, the LOS of M. catarrhalis appears to be fairly well conserved at the antigenic level (Vaneechoutte et al., 1990), thus raising the feasibility of using a portion of the LOS molecule as a vaccine component.
Lastly, the Fimbriae have been suggested as a possible vaccine candidate. Fimbriae apparently play a role in adherence and colonization of mucosal services in some bacteria. Workers in the field have postulated that if antigenically conserved epitopes are expressed on fimbriae and can be identified, then it is possible that antibodies to such epitopes might be useful therapeutically, or that such epitopes can serve as vaccine components.
Despite its recognized virulence potential, little is known about the mechanisms employed by M. catarrhalis in the production of disease or about host. factors governing immunity to this pathogen. An antibody response to M. catarrhalis otitis media has been documented by means of an ELISA system using whole M. catarrhalis cells as antigen and acute and convalescent sera or middle ear fluid as the source of antibody (Leinonen et al., 1981). The development of serum bactericidal antibody during M. catarrhalis infection in adults was first reported nearly a decade ago and this bactericidal activity was shown to be dependent on the classical complement pathway (Chapman et al., 1985). Most recently, it was reported that young children with M. catarrhalis otitis media develop an antibody response in the middle ear but fail to develop systemic antibody in a uniform manner (Faden et al., 1992).
With the rising importance of this pathogen in respiratory tract infections, identification of the surface components of this bacterium involved in virulence expression and immunity is becoming more important. Unfortunately, the lack of a suitable animal model with which to study M. catarrhalis middle ear infections has further hampered such investigations. The relative lack of virulence of this organism for animals has rendered identification of an appropriate model system difficult (Doern, 1986). Attempts to use rodents, including chinchillas, to study middle ear infections caused by M. catarrhalis were unsuccessful, likely because this organism cannot grow or survive in the middle ear of these hosts (Doyle, 1989).
Although various subcomponents of the M. catarrhalis cell have been suggested as places to begin a search for vaccine candidates, there has still been no such candidate identified. No antigenic epitope or epitopes have been shown to induce protective antibodies. Thus, it is clear that there is currently a need to identify M. catarrhalis component(s) which may serve as useful antigens and which can, for example, be employed in the preparation of both passive and active immunotherapeutic reagents such as vaccines. Additionally, once such an antigen or antigens is identified, there is a need for providing methods and compositions which will allow the preparation of these vaccines and quantities that will allow their use on a wide scale basis in therapeutic protocols.